Preincubation with NADPH

Figure 5 Flowchart for initial and possible follow-up CYP inhibition studies. The first box represents the IC50 experiment with and without a 30-minute preincubation with NADPH (the highest concentration of test article is also preincubated for 30 minutes without NADPH). Remaining boxes depict possible outcomes and follow-up experiments. Abbreviations CYP, cytochrome P450 IC50, concentration of inhibition causing 50% inhibition.

Figure 13 Effect of substrate incubation time on the metabolism-dependent inhibition of CYP3A4/5 by mibefradil. Mibefradil (0.01 to 10 pM) was examined as a direct-acting and metabolism-dependent inhibitor with either a 0- or 15-minute preincubation with NADPH followed by either a 5- or 30-minute incubation with testosterone (100 pM). In both cases, the IC50 value after a 15-minute preincubation with NADPH was approximately 0.07 pM. However, the IC50 value for direct inhibition (0-minute preincubation) varied nearly fourfold depending on the length of the substrate incubation, with a longer substrate incubation period diminishing the apparent impact of metabolism-dependent inhibition due to increased inactivation during the substrate incubation.

The FDA-approved and acceptable chemical inhibitors for reaction phenotyping are included in Table 2. Many of the inhibitors listed in Table 2 are metabolism-dependent inhibitors that, in order to inhibit CYP, require preincubation with NADPH-fortified human liver microsomes for 15 minutes or more. In the absence of the metabolism-dependent inhibitor, this preincubation of microsomes with NADPH can result in the partial, spontaneous loss of several CYP enzyme activities (see sec. II.C.7.c). Furthermore, the organic solvents commonly used to dissolve chemical inhibitors can themselves inhibit (or possibly activate) certain CYP enzymes, as discussed in section II.C.4. Therefore, appropriate solvent and preincubation controls should be included in all chemical inhibition experiments. 

Paris BL, Marcum AE, Clarin JR, et al. Effects of common organic solvents on CYP2E1 activity in human liver microsomes effects of order of addition and preincubation with NADPH. Drug Metab Rev 2003 35(suppl 2) 180. 

Figure 6.20. Role of phospholipase D in NADPH oxidase activation. In (a) neimophils were preincubated with [3H]-alkyl-lyso-PAF (5 /iCi/ml) for 60 nun at 37 C. The cells were then washed twice with RPMI 1640 medium and finally resuspended at 2 x 10 cells/ ml in the presence ( ) and absence ( ) of 100 mM ethanol. The cells were then stimulated with 1 pM fMet-Leu-Phe and, at time intervals,aliquots were removed for analysis ofphos-phatidic acid ( ) and phosphatidylethanol ( ) by thin layer chromatography (TLC). In (b), neutrophils were incubated in the presence and absence of 10 mM butanol, and luminol chemiluminescence (10 jUM, final concentration of luminol) was measured after stimulation by 1 jUM fMet-Leu-Phe. Source Experiment of Gordon Lowe and Fiona Watson.

Figure 11 Irreversible inhibition of CYP2A6 by 8-methoxypsoralen at two concentrations with and without a dilution step. The overall design of the experiment is discussed in section II.C.7.b. Panels A and B show the effects of preincubating 8-methoxypsoralen (0.05 and 1.25 pM) for 30 minutes with NADPH-fortified human liver microsomes (0.0125 mg/mL) without a dilution prior to the incubation with substrate (coumarin). Panels B and D show the effects of preincubating 8-methoxypsoralen (0.05 and 1.25 pM) for 30 minutes with NADPH-fortified human liver microsomes (0.3125 mg/mL) with a 25-fold dilution prior to the incubation with substrate (coumarin). Panel E shows the effects of preincubating 8-methoxypsoralen (1.25 pM) for 30 minutes with pooled human liver microsomes (0.0125 mg/mL) in the absence of NADPH without a dilution step prior to the incubation with substrate (coumarin). Inhibition in the latter case is caused by inactivation of CYP2A6 during the substrate incubation step (5 minutes) because it occurs to the same extent in both the 0- and 30-minute preincubation samples.

If a mechanism-based inhibitor is used, a 15- to 30-minute preincubation period with NADPH should be incorporated. 

Typical experimental procedures are as follows The test drug candidate is incubated with pooled human liver microsomes (e.g., 1 mg protein/mL) that were previously preincubated with ABT (1 or 2 mM) for 30 minutes at (37 1)°C in the presence of an NADPH-generating system. Incubations of the drug candidate in the absence of ABT serve as controls. For hepatocytes, suspensions of freshly isolated or cryopreserved hepatocytes (lx 106 cells/ mL) are preincubated with 100-pM ABT for 30 minutes in 0.25 mL of Krebs-Henseleit buffer or Waymouth s medium (without phenol red) supplemented with FBS (4.5%), insulin (5.6 pg/mL), glutamine (3.6 mM), sodium pyruvate (4.5 mM), and dexamethasone (0.9 pM) at the final concentrations indicated. After the preincubation, the drug candidate is added to the incubation and the rate of metabolism of the drug candidate is compared in hepatocytes or microsomes with and without ABT treatment. A marked difference in metabolism caused by ABT is evidence that CYP plays a prominent role in the metabolism of the drug candidate. 

In our normal assays with R-5-P + ATP we do not have these conditions (FIG-1) and even after preincubation with RuBP and ATP the dark extracts showed 64% activity as compared to 79% obtained with R-5-P + ATP (TABLE-1). 2. We have not added NADPH in these assays therefore the question of role of the NADPH dependent chloroplastic protein does not arise. It is most likely that freshly synthesized RuBP in the multienzyme complex of RuBP carboxylase, kinase and isomerase must be getting channelled to the active site of RuBP carboxylase, replacing the inhibitor and there by reactivating the dark inactivated enzyme. Since phosphoribulokinase has been shown to be activated by light the above mechanism of reactivation of dark-inhibited RuBP carboxylase would be important Jii vivo. 

Fig. 2. Inactivation of estradiol 17/3-dehydrogenase by 16a-bromoacetoxyestra-diol 3-methyl ether and protection against enzyme inactivation by cofactor or substrate. Aliquots were assayed each point is the mean of four determinations. Enzyme (1.4 nmoles) was preincubated with 16o -bromoacetoxyestradiol 3-methyl ether (0.21 /tmole) in 15 ml of buffer A, 25°, pH 7.0 containing 20% ethanol, (O) 16a-bromo-acetoxyestradiol 3-methyl ether (051 nmole) and NADH (0.42 /tmole), (A) 16 -bromoacetoxyestradiol 3-methyl ether (051 /amole) and NADPH (0.42 /tmole) ( ) lOa-bromoacetoxyestradiol 3-methyl ether (051 /imole) and estradiol (051 /imole) ( ) and control or with bromoacetic acid (0.42 mole) (A). From C. C. Chin and J. C. Warren, J. Biol. Chem. 250, 7682 (1975).

Assayed by coupling with the next reaction and following NADPH oxidation spectrophotometrically. SH-enzyme inhibited by iodoacetamide, etc. Protected by preincubation with acetyl-ACP not malonyl-ACP... 

After removal of free Emulgen 913 from partially purified hepatic cytochrome P-448 of DBA-treated male skates an active mixed-function oxidase system was reconstituted by preincubating the cytochrome with purified rabbit hepatic NADPH-cytochrome o... 

Time-dependent inhibition should be examined. A 30-minute preincubation (i.e., with nicotinamide adenine dinucleotide phosphate (NADPH) enzyme, and drug candidate prior to addition of the probe substrate) is recommended. 

It is recommended that time-dependent inhibition be examined when deemed appropriate. Time-dependent inhibition should be examined with and without NADPH over an inhibitor concentration range of 1- to 10-fold the clinically relevant plasma concentrations. Various preincubation time points, such as 0, 15, 30, 45, and 60 minutes, should be utilized along with at least a 10-fold dilution step prior to the substrate incubation. 

CYP2E1 was additionally coexpressed with the human cytochrome b-5. Microsomal protein, 500 (xg, was resuspended in 500 pi of 20 mM Tris/HCl buffer (pH = 7.4). Study into perazine metabolism in Supersomes was carried out at the neuroleptic concentration of 750 11M (3 Km) allowing to reach the velocity of reaction of about Vmax to show the maximum ability of cDNA expressed enzyme to metabolize perazine. After 3-min preincubation at 37 °C, the reaction was initiated by adding NADPH to a final concentration of 0.1 mM. After 2 h incubation, the reaction was stopped by adding 200 pi of methanol. Perazine and its metabolites were analyzed by HPLC. 

The presence of two coenzyme-binding sites is unexpected since they cannot be inferred solely from the crystal structure of CPR. Kinetic studies with wild t) e and W676H CPR at different concentrations of NADPH have, however, provided further support for the existence of two sites The rate of flavin reduction in the isolated FAD domain and CPR increases as NADPH is decreased from molar excess to stoichiometric concentrations. At stoichiometric concentration, the second noncatalytic site is predominantly vacant and the partial inhibition on the rate of flavin reduction from the catalytic site is therefore relieved (Figure 4.9). Occupation of the noncatalytic site occurs at NADPH concentrations in excess of the enzyme concentration, and impairs NADP" " release from the catalytic site. This in turn partially inhibits flavin reduction, the rate of which is gated by NADP release. Preincubation of the enzyme with a stoichiometric amount of adenosine 2, 5 -diphosphate does not lead to inhibition of the flavin reduction rate. We infer that the binding of adenosine 2, 5 -diphosphate prevents NADPH from binding to the noncatalytic site. This observation also suggests that it is the nicotinamide-ribose-phosphate portion of NADPH bound at the second site that hinders NADP" release from the catalytic site. Clearly, these new... 

GSH (1 mM), and NADPH (1 mM) in potassium phosphate buffer (100 mM, pH 7.4) for 30 min [24], The total incubation volume was 2 mL. The incubation reactions were initiated by the addition of an NADPH solution after a 3-min preincubation, and were stopped by the addition of 300 pL of trichloroacetic acid (10%). After centrifugation (13,000 rpm for 10 min), supernatants were loaded onto SPE cartridges (Oasis extraction cartridges, Waters Corp., Milford, MA). The cartridges were washed with 1 mL of water and then eluted with 2 mL of methanol. The methanol fractions were dried and reconstituted with 200 pL of a water/acetonitrile mixture (v/v, 95 5). Aliquots (20 pL) of the reconstituted solutions were injected into LC-MS/MS. 

Because ATP involvement may be required for the endogenous synthesis of an acyl primer, such as acyl-CoA, we examined the effects of preincubation of leek or B.napus microsomes with [ " CJfree fatty acids on elongation rates in the presence or absence of ATP. Preincubation did not have any significant effect on the elongation rate, and we did not observe accumulation of acyl-CoA during preincubation and before the elongation reaction was initiated by the addition of malonyl-CoA and NADPH (data not shown). 

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